Journal:Acta Cryst D:S205979832001517X
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| - | <StructureSection load='' size='450' side='right' scene=' | + | <StructureSection load='' size='450' side='right' scene='86/869943/Cv/1' caption=''> |
===An engineered disulfide bridge traps and validates an outward-facing conformation in a bile acid transporter=== | ===An engineered disulfide bridge traps and validates an outward-facing conformation in a bile acid transporter=== | ||
<big>Xiaodong Wang, Ying Lyu, Yujia Ji, Ziyi Sun and Xiaoming Zhou</big> <ref>doi: 10.1107/S205979832001517X</ref> | <big>Xiaodong Wang, Ying Lyu, Yujia Ji, Ziyi Sun and Xiaoming Zhou</big> <ref>doi: 10.1107/S205979832001517X</ref> | ||
<hr/> | <hr/> | ||
<b>Molecular Tour</b><br> | <b>Molecular Tour</b><br> | ||
| + | Apical sodium-dependent bile acid transporter (ASBT) is a membrane transporter protein which recycles bile acids from small intestine into enterocytes. It is a potential drug target for treating several metabolic diseases including type 2 diabetes. To date, high-resolution structures are only available for two bacterial ASBT proteins, which provide structural information to help understand how a bile acid is transported by ASBT. To do so, a minimum of two ASBT conformational states have to be obtained, one facing outside of the cell and the other facing inside. However, currently the only outward-facing ASBT structure is obtained with a severely crippled ASBT protein, whose normal functions such as binding and transport of bile acids are nearly lost. Therefore, the outward-facing state of ASBT needs validation. | ||
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| + | In this study, a bacterial ASBT, called ASBT<sub>Yf</sub>, is engineered to have its intracellular parts connected by a disulfide bond, so that it will be trapped in an outward-facing state. Before this engineered ASBT<sub>Yf</sub> is trapped, it folds and moves like the wild-type protein. More importantly, it remains functional since it binds bile acids as normal. Then after the conformational trap, this ASBT<sub>Yf</sub> is found to open outwardly in its structure. In other words, a wild-type-like ASBT protein is trapped in a state facing outside of the cell, demonstrating that this outward-facing state is of physiological relevance. Meanwhile, a low-affinity ligand-like molecule, citrate, binds to the substrate-binding site in the trapped outward-facing ASBT structure, further indicating that the trapped ASBT protein retains its functionality. These data validate a physiological outward-facing state in ASBT, and advance out understanding toward its transport mechanism. | ||
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| + | <scene name='86/869943/Cv/6'>ASBT structure trapped in the outward-facing state by an engineered disulfide bond</scene> (PDB entry [[6lh1]]). Solvent can access the central binding pocket from outside of the cell. | ||
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| + | *<scene name='86/869943/Cv/8'>Close-up of engineered disulfide bond</scene>. | ||
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| + | <scene name='86/869943/Cv/9'>A ligand-like molecule, citrate, binds to the central binding pocket in the outward-facing ASBT</scene> (PDB entry [[6lh1]]), indicating this protein retains functionality. | ||
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| + | '''PDB references:''' ASBTYf-Pair1Linked, Y113C/P190C mutant after disulfide cross-linking, [[6lh1]]; ASBTYf-Pair1Free, Y113C/P190C mutant before disulfide cross-linking, [[7cyg]]; ASBTYf-Pair2Free, V110C/I197C mutant before disulfide cross-linking, [[7cyk]]. | ||
<b>References</b><br> | <b>References</b><br> | ||
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